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Abstract:

CK1 has a potential phosphorylation dependant 14-3-3 binding site within the same region previously shown to be the interaction site for centaurin-α1 and the aim of this investigation was to examine the possible interaction. 14-3-3 was found to associate with various CK1 isoforms, with CK1α being studied further to reveal an interaction through serine residues 218 and 242 in a phosphorylation dependant manner, in vivo. Centaurin-αl was found to interact with a region corresponding to residues 213-226, only if S218 was in a dephosphorylated state, suggesting a possible regulatory mechanism. Mutagenesis of CK1α suggests that S242 is a high affinity binding site for 14-3-3, with S218 being of lower affinity. Investigations to identify possible kinase(s) responsible for phosphorylation of CK1 showed that stimulation of PKA can increase CK1α:14-3-3 association in cells, but PKA does not appear to phosphorylate CK1α in vitro. As phosphorylation of 14-3-3 itself is an important regulatory mechanism, attempts were made to produce antibodies to phosphorylated S185 and S233 on 14-3-3. A phospho-specific antibody to S185 was successful, but antibodies to α-phospho-S233 had no preference to the phosphorylation state of 14-3-3, although they were of high selectivity and affinity for 14-3-3 isoforms. The effect of S185 phosphorylation on the interaction with signalling molecules was studied. The kinase BCR (Breakpoint cluster region) is an important, but poorly understood protein that has been shown to associate with and phosphorylated 14-3-3. Investigations showed that BCR phosphorylates 14-3-3 on Ser233 in vitro. Additionally, BCR is shown to associate with another two isoforms of 14-3-3 both in vitro and in vivo. However BCR selectively phosphorylates 14-3-3 τ more that ζ in contrast to CK1.